Phosphorothioic triamide, N-propyl-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May - June 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

April 24, 2002

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(from the competent authority) Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of N-butylphosphorothioic triamide and N-propylphosphorothioic triamide

EC Number:

700-457-2

Molecular formula:

Unspecified

IUPAC Name:

Reaction mass of N-butylphosphorothioic triamide and N-propylphosphorothioic triamide

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 8712 / 056
- Purity: 99.6 g / 100 g

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the vehicle: The stability of the test substance in the vehicle was determined indirectly by the concentration control or homogeneity analysis. For this purpose, the samples taken were stored at room temperature over the maximum duration of the application period and were subsequently deep-frozen. Afterwards, these samples were analyzed.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance preparation was produced on a weight per weight basis shortly before the application by stirring with a magnetic stirrer. The homogeneity of the test substance preparation during application was provided by stirring with a magnetic stirrer.

FORM OF APPLICATION
Suspension

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: 6 - 12 weeks
- Weight at study initiation: 19.1 - 22.1 g
- Housing: single housing in fully air-conditioned rooms
- Diet (e.g. ad libitum): Kliba-Labordiät, Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 7 days before the first test substance application

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): fully air-conditioned
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

- Control group 1: Vehicle (MEK)
- Test group 2: Test substance 3 % in MEK
- Test group 3: Test substance 10 % in MEK
- Test group 4: Test substance 50 % MEK

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
The selection of the concentrations took into account available information on the chemical / physical properties, the composition and on primary irritation / corrosion potential of the test substance. In addition the results of a pretest were considered, which did not show increased ear weights and lymph node weights after application of a 50 % test substance preparation in MEK (methyl ethyl ketone).
Higher concentrations were not tested in order to avoid ear skin irritation and because the 50 % concentration was a suspension and well above the effect level of mild to moderate skin sensitizers, leading to a sufficiently stringent conduct of the study.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Randomization: Prior to first application, the animals were distributed to the individual groups, received their animal numbers and were allocated to the respective cages according to the randomization instructions of "Nijenhuis, A. and Wilf, H.S.: Combinatorial Algorithms, Academic Press, New York, San Francisco, London, 1978, pp. 62 - 64".

TREATMENT PREPARATION AND ADMINISTRATION:
- Form of application: Epicutaneous application is simulating dermal contact with the compound which is possible to occur under practical use conditions.
- Application volume: 25 µL per ear
- Site of application: Dorsal part of both ears
- Frequency of application: 3 consecutive applications (day 0 - day 2) to the same application site

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The stimulation indices of cell count, 3H-thymidine incorporation, lymph node weight and ear weight were calculated as the ratio of the test group values for these parameters divided by those of the vehicle control group.

In order to reveal a possible induction of sensitization, the response in the draining lymph node after epicutaneous application of several concentrations of the test substance to the skin of the ear backs is determined. The parameters used to characterize the response are lymph node cell count, 3H-thymidine incorporation into the lymph node cells and to a certain extent lymph node weight. Because not only sensitization induction but also irritation of the ear skin by the test substance may induce lymph node responses, the weight of ear punches taken from the area of test substance application is determined as a parameter for inflammatory ear swelling serving as an indicator for the irritant action of the test substance.
The increase SI of cell count by a factor of ≥ 1.5 and / or of 3H-thymidine incorporation by a factor of ≥ 3 as compared to the concurrent vehicle control group is generally considered as indicating a sensitizing potential of a test substance.
If applicable, the estimated concentration leading to the respective SI values were calculated by linear or semi-logarithmical regression between the data points directly below and above the SI if possible or using the two nearest points below or above the SI.

Results and discussion

Positive control results:

The sensitivity of mice and the reliability of experimental techniques was assessed regularly using a known sensitizer.
Positive results were consistently obtained over the years using several variations of the methods and different vehicles.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks:

Cell count

Value:

1

Test group / Remarks:

Control group 1, vehicle MEK

Results 2

Parameter:

SI

Remarks:

Cell count

Value:

0.93

Test group / Remarks:

Test group 2, 3 % in MEK

Results 3

Parameter:

SI

Remarks:

Cell count

Value:

1.11

Test group / Remarks:

Test group 3, 10 % in MEK

Results 4

Parameter:

SI

Remarks:

Cell count

Value:

1.09

Test group / Remarks:

Test group 4, 50 % in MEK

Results 5

Parameter:

SI

Remarks:

3H-thymidine incorporation

Value:

1

Test group / Remarks:

Control group 1, vehicle MEK

Results 6

Parameter:

SI

Remarks:

3H-thymidine incorporation

Value:

1.25

Test group / Remarks:

Test group 2, 3 % in MEK

Results 7

Parameter:

SI

Remarks:

3H-thymidine incorporation

Value:

0.67

Test group / Remarks:

Test group 3, 10 % in MEK

Results 8

Parameter:

SI

Remarks:

3H-thymidine incorporation

Value:

2.3

Test group / Remarks:

Test group 4, 50 % in MEK

Results 9

Parameter:

SI

Remarks:

Lymph node weight

Value:

1

Test group / Remarks:

Control group 1, vehicle MEK

Results 10

Parameter:

SI

Remarks:

Lymph node weight

Value:

0.98

Test group / Remarks:

Test group 2, 3 % in MEK

Results 11

Parameter:

SI

Remarks:

Lymph node weight

Value:

1.05

Test group / Remarks:

Test group 3, 10 % in MEK

Results 12

Parameter:

SI

Remarks:

Lymph node weight

Value:

1.14

Test group / Remarks:

Test group 4, 50 % in MEK

Results 13

Parameter:

SI

Remarks:

Ear weight

Value:

1

Test group / Remarks:

Control group 1, vehicle MEK

Results 14

Parameter:

SI

Remarks:

Ear weight

Value:

1.05

Test group / Remarks:

Test group 2, 3 % in MEK

Results 15

Parameter:

SI

Remarks:

Ear weight

Value:

1.11

Test group / Remarks:

Test group 3, 10 % in MEK

Results 16

Parameter:

SI

Remarks:

Ear weight

Value:

1.05

Test group / Remarks:

Test group 4, 50 % in MEK

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
When applied as 3 %, 10 % and 50 % preparations in MEK, the test substance did not induce a biologically relevant response (increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts.
There was no relevant increase in lymph node weights, as well.
Concomitantly, the concentration-independent increase of 3H-thymidine incorporation into the cells was not biologically relevant (increase above the cut off stimulation index of 3) at these concentrations. The low value found for the animals treated with the 10 % concentration does not correlate to the cell count and is considered to be an incidental finding.
The test substance preparations caused minimal concentration-independent increase in ear weights.

CLINICAL OBSERVATIONS: No abnormalities were observed during general observation.

BODY WEIGHTS: The expected body weight gain was generally observed in the course of the study.

Any other information on results incl. tables

Table 1: Cell counts

Test group	Treatment	Counts / lymph node pair	Stimulation index^1
1	Vehicle MEK	5,889,333	1.00
2	3 % in MEK	5,498,667	0.93
3^*	10 % in MEK	6,528,333	1.11
4	50 % in MEK	6,413,333	1.09

¹: test group x / test group 1 (vehicle control)

^*: calculation on basis of 4 animals, as one animal died during³H-thymidine injection

Table 2: 3H-thymidine incorporation

Test group	Treatment	DPM / Lymph node pair	Stimulation index^1
1	Vehicle MEK	327.1	1.00
2	3 % in MEK	407.6	1.25
3^*	10 % in MEK	219.5	0.67
4	50 % in MEK	753.7	2.30

¹: test group x / test group 1 (vehicle control)

^*: calculation on basis of 4 animals, as one animal died during³H-thymidine injection

Table 3: Lymph node weight

Test group	Treatment	mg / Lymph node pair	Stimulation index^1
1	Vehicle MEK	4.3	1.00
2	3 % in MEK	4.2	0.98
3^*	10 % in MEK	4.5	1.05
4	50 % in MEK	4.9	1.14

¹: test group x / test group 1 (vehicle control)

^*: calculation on basis of 4 animals, as one animal died during³H-thymidine injection

Table 4: Ear weight: test group mean values and stimulation indices

Test group	Treatment	mg / animal	Stimulation index^1
1	Vehicle MEK	30.5	1.00
2	3 % in MEK	32.0	1.05
3^*	10 % in MEK	33.9	1.11
4	50 % in MEK	32.0	1.05

¹: test group x / test group 1 (vehicle control)

^*: calculation on basis of 4 animals, as one animal died during³H-thymidine injection

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

No signs of systemic toxicity were noticed.
When applied as 3 %, 10 % and 50 % preparation in MEK, the test substance did not induce a biologically relevant response (increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts.
There was no relevant increase in lymph node weights, as well.
Concomitantly, the concentration-independent increase of 3H-thymidine incorporation into the cells was not biologically relevant (increase above the cut off stimulation index of 3) at these concentrations.
The concentration-independent minimal increases in ear weights might be interpreted as signs of very mild ear skin irritation.
Thus it is concluded that the test substance does not show a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.

Executive summary:

The skin sensitizing potential of the test substance was assessed using the radioactive Murine Local Lymph Node Assay. The assay simulates the induction phase for skin sensitization in mice. It determines the response of the auricular lymph nodes on repeated application of the test substance to the dorsal skin of the ears.

Groups of 5 female CBA/J mice each were treated with 3 %, 10 % and 50 % w/w preparations of the test substance in MEK (methyl ethyl ketone) or with the vehicle alone. Each test animal was applied with 25 µL per ear of the respective test substance preparation to the dorsum of both ears for three consecutive days. The control group was treated with 25 µL per ear of the vehicle alone.

Three days after the last application the mice were injected intravenously with 20 µCi of 3H-thymidine in 250 µL of sterile saline into a tail vein. About 5 hours after the 3H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. The weights of each animal's pooled lymph nodes were determined. Thereafter lymph nodes were pooled group wise and further evaluated by measuring their cellular content and 3H-thymidine incorporation into the lymph node cells (indicators of cell proliferation). Moreover, a defined area with a diameter of 0.8 cm was punched out of the apical part of each ear and for each test group the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation.

The stimulation indices (fold of change as compared to the vehicle control) for cell count, 3H-thymidine incorporation, lymph node weight and ear weight are summarized for each test group in the table below.

Test group	Treatment	Cell Count Stimulation Index	^3H-thymidine incorporation Stimulation Index	Lymph Node Weight Stimulation Index	Ear Weight Stimulation Index
1	Vehicle MEK	1.00	1.00	1.00	1.00
2	3 % in MEK	0.93	1.25	0.98	1.05
3^*	10 % in MEK	1.11	0.67	1.05	1.11
4	50 % in MEK	1.09	2.30	1.14	1.05

^*: Calculation on basis of 4 animals, as one animal died during³H-thymidine injection

No signs of systemic toxicity were noticed.

When applied as 3 %, 10 % and 50 % preparations in MEK, the test substance did not induce a biologically relevant response (increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts.

There was no relevant increase in lymph node weights, as well.

Concomitantly, the concentration-independent increase of 3H-thymidine incorporation into the cells was not biologically relevant (increase above the cut off stimulation index of 3) at these concentrations.

The concentration-independent minimal increases in ear weights might be interpreted as signs of very mild ear skin irritation.

Thus it it concluded that the test substance does not show a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.